Caruncular Endometrium Research Articles

In vivo progestin treatments inhibit nitric oxide and endothelin-1-induced bovine endometrial prostaglandin (PG) E (PGE) secretion in vitro

Synchronization of estrus with progestins in cows has been reported to inhibit nitric oxide (NO) and endothelin-1 (ET-1)-stimulated bovine luteal PGE secretion without affecting prostaglandin F 2α (PGF 2α) secretion in vitro [Weems YS, Randel RD, Tatman S, Lewis A, Neuendorff DA, Weems CW. Does estrous synchronization affect corpus luteum (CL) function? Prostaglandins Other Lipid Mediat 2004;74:45–59]. Two experiments were conducted to determine the effects of NO donors, endothelin-1 (ET-1), and NO synthase (NOS) inhibitors on bovine caruncular endometrial secretion of PGE and PGF 2α in vitro. In Experiment 1, estrus was synchronized in Brahman cows with Synchromate-B ear implants, which contained the synthetic progestin norgestamet. Days 14–15 caruncular endometrial slices were weighed, diced, and incubated in vitro with treatments. Treatments (100 ng/ml) were: Vehicle (control), l-NAME (NOS inhibitor), l-NMMA (NOS inhibitor), DETA (control), DETA-NONOate (NO donor), sodium nitroprusside (NO donor), or ET-1. In Experiment 2, estrus was synchronized in Brahman cows with either Lutalyse (PGF 2α) or a controlled intravaginal drug releasing device (CIDR-containing progesterone) or estrus was not synchronized. Days 14–15 caruncular endometrial slices were weighed, diced, and incubated in vitro with treatments. Treatments (100 ng/ml) were: vehicle, l-NAME, l-NMMA, DETA, DETA-NONOate, sodium nitroprusside, SNAP (NO donor) or ET-1. Tissues were incubated in M-199 for 1 h without treatments and with treatments for 4 and 8 h in both experiments. Media were analyzed for concentrations of PGE and PGF 2α by radioimmunoassay (RIA). Hormone data in Experiments 1 and 2 were analyzed by 2 × 7 and 3 × 2 × 8 factorial design for ANOVA, respectively. Concentrations of PGE and PGF 2α in media increased ( P ≤ 0.05) from 4 to 8 h regardless of treatment group in Experiment 1, but did not differ ( P ≥ 0.05) among treatments. In Experiment 2, concentrations of PGE and PGF 2α increased ( P ≤ 0.05) with time in all treatment groups of all three synchronization regimens. DETA-NONOate, SNAP, and sodium nitroprusside (NO donors) and ET-1 increased caruncular endometrial ( P ≤ 0.05) secretion of PGE 2 in unsynchronized and Lutalyse synchronized cows, but not when estrus was synchronized with a CIDR ( P ≥ 0.05). No treatment increased ( P ≥ 0.05) PGF 2α in any synchronization regimen. It is concluded that norgestamet in Synchromate-B ear implants or progesterone in a CIDR alters NO or ET-1-induced secretion of PGE by bovine caruncular endometrium and could interfere with implantation by altering the PGE:PGF 2α ratio resulting in increased embryonic losses during early pregnancy.

The effect of dietary supplementation with linoleic acid to late gestation ewes on the fatty acid composition of maternal and fetal plasma and tissues and the synthetic capacity of the placenta for 2-series prostaglandins

Linoleic acid (18:2 n-6) is metabolised to arachidonic acid (20:4 n-6), the precursor for 2-series prostaglandins (PGs). Increased consumption of 18:2 n-6 during pregnancy may thus modify PG synthesis during labour. We have investigated whether increased 18:2 n-6 composition during gestation altered the fatty acid consumption and PG synthesis of maternal and fetal tissues in the sheep. Ewes were fed a control diet or a diet providing 40% more 18:2 n-6 from 96 days gestation. Half of each group received dexamethasone on day 136 to up-regulate the PG synthetic pathways promoting parturition. Maternal and fetal tissues were collected at 138 days. The 18:2 n-6 diet significantly increased the 20:4 n-6 content of maternal plasma, fetal plasma and allantochorion (51–81%) phosphatidylcholine, and fetal liver (40%) and maternal caruncular endometrium (57%) phosphatidylethanolamine. Increased 18:2 n-6 intake increased production of PGF 2α and PGE 2 in all placental tissues (maternal caruncular and intercaruncular endometrium and fetal allantochorion) by 23–98%, whereas dexamethasone increased it by 32–142%. This suggests that consumption of an 18:2 n-6-enriched diet in late pregnancy enhanced placental PG production by increasing the supply of 20:4 n-6. Variations in the extent to which the diet altered the polyunsaturated fatty acid (PUFA) content of the different tissues indicated complex interactions between nutrient availability and metabolic adaptation.

Effect of fish meal supplementation on plasma and endometrial fatty acid composition in nonlactating beef cows.

Seven nonlactating mature Angus cows (4 to 10 yr old) were used to examine the effects of fish meal supplementation on plasma and endometrial fatty acid composition. Cows were fed a corn silage-based diet supplemented with either fish meal, a rich source of the n-3 fatty acids, eicosapentaenoate and docosahexaenoate (n = 3; 5.1% of dietary DM), or corn gluten meal (n = 4; 8.5% of dietary DM) for approximately 64 d. Cows were given 25 mg of PGF2alpha (i.m.) on d 11 and 25 of supplementation to synchronize estrous cycles. On d 18 postestrus of the second estrous cycle, cows were slaughtered, and caruncular endometrium was dissected from uteri immediately after slaughter. Jugular blood samples were collected immediately before supplementation was initiated (d 0) and at 7-d intervals for 35 d of the study. Plasma eicosapentaenoic and docosahexaenoic acids did not differ between treatment groups on d 0 (P > 0.10); however, these fatty acids were greater in cows supplemented with fish meal over the first 35 d of supplementation compared with cows supplemented with corn gluten meal (P < 0.05). Endometrial docosahexaenoic acid did not differ (P = 0.12), whereas eicosapentaenoic acid was greater (P < 0.05) in cows supplemented with fish meal than in cows supplemented with corn gluten meal. These results indicate that dietary fish meal alters plasma and endometrial n-3 fatty acid composition in beef cows.

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium during early pregnancy

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16–17, 20–21, 30–36, 48–49 and 74–140) and during the estrous cycle (Days 13–14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48–49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48–49 and 74–140 of pregnancy were greater than at Days 13–14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74–140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.

Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E 2 (PGE 2) and F 2α (PGF 2α) and progesterone in vitro

The objective of this experiment was to determine the effect of AA, LH, or PSPB on secretion of PGE 2, PGF 2α, or progesterone by ovine caruncular endometrium of the estrous cycle or placental tissue of pregnancy in vitro. Ovine caruncular endometrium of the estrous cycle (days 8, 11, 13, and 15) or caruncular/placental tissue on days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90 postbreeding were incubated in vitro with vehicle, AA, LH, or PSPB in M-199 for 4 and 8 h. Secretion of PGF 2α by caruncular endometrium of non-bred ewes on days 13 and 15 and by caruncular/placental tissue of bred ewes on days 13, 15, 20, 30, and 40 was increased ( P≤0.05) when incubated with vehicle and declined ( P≤0.05) after day-40 in bred ewes. Secretion of PGF 2α by day-15 caruncular endometrium of non-bred ewes and bred ewes was increased ( P≤0.05) by AA on days 13 and 15 and by LH on day-15. Secretion of PGF 2α by caruncular/placental tissue from bred ewes was increased ( P≤0.05) by AA on days 13, 15, 20, 30, and 40 and by LH on days 15, 20, 30, and 40, after which the response decreased ( P≤0.05). Secretion of PGF 2α by caruncular endometrium of non-bred ewes during the estrous cycle or by caruncular/placental tissue of bred ewes during the first trimester was not affected by PSPB ( P≥0.05). Secretion of PGE 2 by caruncular endometrium of non-bred ewes did not change ( P≥0.05) and was increased ( P≤0.05) by caruncular/placental tissue on days 13–90 from bred ewes when incubated with vehicle. Secretion of PGE 2 by endometrium from non-bred ewes was not affected ( P≥0.05) by AA, LH, or PSPB, but was increased ( P≤0.05) by AA or LH on days 13–50 and by PSPB on days 60 and 90 when incubated with caruncular/placental tissue from bred ewes. Secretion of progesterone by placental tissue of bred ewes increased ( P≤0.05) on day-50 and continued to increase through day-90. In summary, uterine/placental tissue secretion of PGF 2α is not reduced until the end of the first trimester of pregnancy in ewes. In addition, LH appears to play a role in luteolysis of non-bred ewes by stimulating caruncular endometrial secretion of PGF 2α and on day-5 postbreeding to prevent luteolysis during early pregnancy by stimulating caruncular/placental secretion of PGE 2 throughout the first trimester of pregnancy in sheep. Secretion of PGE 2 by caruncular/placental tissue after day-50 of pregnancy appears to be regulated by PSPB, not LH.

Expression of fertilin and CD9 in bovine trophoblast and endometrium during implantation.

The superficial placentation of cattle involves the development of fetal binucleate cells that arise from the chorion and migrate between adjacent cell tight junctions to fuse with maternal epithelium. Thus, the temporal and spatial patterns of expression of the cell migration, adhesion, and fusion molecules fertilin and CD9 were investigated in bovine trophoblast and endometrium. Bovine fertilin alpha and fertilin beta messenger RNA sequences were amplified by reverse transcriptase-polymerase chain reaction in testis (positive control), peri-implantation (Days 18, 19, and 21), and postimplantation (Days 35-40) trophoblast RNA, but not in caruncular endometrium (Day 40). Northern blot analysis indicated that the transcript hybridizing to fertilin alpha in trophoblast RNA was approximately 4.0 kilobases (kb), whereas in testis, 2 transcripts of approximately 3.3 and 3.8 kb were indicated. The transcript hybridizing to the fertilin beta probe was also larger in trophoblast than in testis ( approximately 3.8 vs. 2.4 kb, respectively). In situ hybridization revealed that fertilin beta mRNA was expressed by trophoblast cells, including binucleate cells. Immunohistochemical study of CD9, a member of the transmembrane-4-superfamily which is thought to be involved in sperm-egg fusion, showed that CD9 was present on the apical surface of uterine epithelium and in a subpopulation of binucleate cells of the trophoblast. Immunoprecipitation followed by Western blot analysis showed association between CD9 and integrin alpha3 in endometrium. The results support the hypothesis that fertilin and CD9 are involved in bovine binucleate cell migration and fusion.

Cellular mechanisms by which oxytocin mediates ovine endometrial prostaglandin F2alpha synthesis: role of G(i) proteins and mitogen-activated protein kinases.

Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2alpha synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2alpha synthesis. The objective of experiment 1 was to determine whether G(i) proteins mediate oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of G(i) proteins, had no effect on the ability of oxytocin to induce PGF2alpha synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF(2alpha) synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF(2alpha synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to G(i) proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2alpha synthesis in ovine endometrium.

PrP-C and PrP-Sc at the Fetal-Maternal Interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.

Effect of oxytocin on expression of cytosolic phospholipase A 2 mRNA and protein in ovine endometrial tissue in vivo

The induction of endometrial prostaglandin (PG) F 2α synthesis by oxytocin is dependent upon activation of phospholipase (PL) A 2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF 2α synthesis by inducing synthesis of cytosolic PLA 2 (cPLA 2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F 2α (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA 2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA 2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA 2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF 2α synthesis by activating cPLA 2 protein that is already present in an inactive form.

Expression of a cytosolic phospholipase A2 by ovine endometrium on days 11-14 of a simulated oestrous cycle.

Oxytocin stimulates the synthesis and secretion of PGF2 alpha from uterine tissues in vivo and in vitro late in the ovine oestrous cycle. The synthesis of eicosanoids is dependent upon the availability of free arachidonic acid which is released through the activity of arachidonate releasing phospholipases. In the present study, the following hypothesis was tested: the ovine endometrium expresses a cytosolic phospholipase A2 (cPLA2) and expression or activity of cPLA2 increases as uterine secretory responsiveness to oxytocin develops late in the oestrous cycle. Endometrial tissue was collected from cyclic ewes on day 15 of the oestrous cycle for the preparation of tissue homogenates and isolation of mRNA to determine whether ovine endometrium expressed a cPLA2. A 110 kDa band was detected by western blotting, indicating the presence of a putative ovine cPLA2. A 834 bp fragment of the ovine cPLA2 shared 87% homology with human and mouse cDNA, and northern blot hybridization analysis indicated a single 3.4 kb transcript. A total of 20 ewes were ovariectomized and treated with progesterone and oestrogen to simulate the oestrous cycle to determine whether the expression or activity of ovine cPLA2 changed during the onset of uterine secretory responsiveness to oxytocin in vivo. On days 11-14 (n = 5 per day) of a simulated oestrous cycle, caruncular endometrium was evaluated for expression of ovine cPLA2 mRNA and protein and the synthesis of PGF2 alpha in response to melittin (a potent stimulator of PLA2 activity). Immunoreactive cPLA2 and cPLA2 mRNA were observed on all days and did not increase during the development of uterine responsiveness to oxytocin in vivo. Similarly, melittin increased the synthesis of PGF2 alpha irrespective of day, indicating the presence of a functional cPLA2 on all days examined. These data indicate that the ovine endometrium expresses a functional cPLA2 and that ample concentrations of cPLA2 are present by day 11 of a simulated oestrous cycle.

Accumulation of cyclooxygenase-2 gene transcripts in uterine tissues of pregnant and parturient cows: stimulation by oxytocin.

Cyclooxygenase 1 and 2 (COX-1 and COX-2) mRNA were measured by ribonuclease protection assays in total RNA extracted from intercaruncular and caruncular endometrium, myometrium, cotyledons, and cervical mucosa of pregnant cows. Tissues were obtained at gestational ages of 150 days and 275 days and at term not in labor, at term in labor, and 6-12 h postpartum. Additionally, the effect of oxytocin (OT) on COX-2 expression was determined in intercaruncular endometrium of six third-trimester cows (between 230 and 270 days of pregnancy), three of which were injected with OT (200 IU) and three with saline 2 h before tissues were harvested. Prostaglandin F2alpha (PGF2alpha) metabolite was measured in plasma samples taken at 15-min intervals before and after the injections. Results showed that COX-2 mRNA was expressed in every type of tissue examined, although in different concentrations and beginning at different stages. Other than in seminal vesicular and prostate glands used as positive controls, low concentrations of COX-1 mRNA were detected only in myometrium and caruncles. Cotyledons had the highest concentration of COX-2 transcripts at all stages studied. Caruncles had about half the concentration of COX-2 transcripts that was seen in cotyledons, and on Day 150 even less. COX-2 mRNA expression in both tissues increased with advancing gestation, but there was no difference between samples from term-no-labor and term-in-labor cows. COX-2 mRNA concentrations in endometrium and myometrium were low; they varied randomly during pregnancy with no significant increase until postpartum, when COX-2 transcripts in endometrium had increased severalfold whereas those in myometrium were similar to values before parturition. Cervical mucosa expressed COX-2 mRNA weakly until term but had increased markedly at parturition. Injection of 200 IU of OT induced a substantial increase in endometrial COX-2 mRNA concentration within 2 h; this was associated with linearly increasing plasma concentrations of 13, 14-hydroxy-15-keto-prostaglandin F2alpha, which were still rising at termination of the experiment. The results suggest that endogenous OT is a major factor in induction of COX-2 expression and PGF2alpha release at term and during parturition in cows.

Blastocyst Development and Conceptus Sex Selection in Red DeerCervus elaphus:Studies of a Free-Living Population on the Isle of Rum

Skewing of the sex ratio at birth occurs in red deer in response to dominance status, with dominant hinds giving birth to a higher proportion of male calves than subordinates. To investigate the physiological basis for this phenomenon, reproductive tracts were collected from red deer during a cull for management purposes carried out on the Island of Rum, Scotland. Blastocysts were flushed from the uterus and sexed by polymerase chain reaction using Y chromosome-specific primers. Concentrations of interferon (measured as antiviral activity) in uterine flushings, of oxytocin receptors in endometrium, and of progesterone in jugular venous blood were measured, and ovarian morphology was recorded. Times of mating were determined retrospectively from calving dates observed during the following spring. Changes in uterine and fetal weights and sizes confirmed the degree of reproductive synchrony. Intervals between stages of blastocyst development (spherical, tubular, filamentous, and attached) derived from the observed incidence of each form showed that approximate times of blastocyst elongation and attachment were 13 and 30 days after conception, respectively. Hinds carrying male blastocysts were in better body condition (higher kidney fat weights,P=0.025) than those carrying females. Interferon was detectable in uterine flushings from 1 of 7 hinds carrying early filamentous blastocysts and 5 of 12 hinds carrying late filamentous blastocysts, but in no case where the blastocysts were male (P=0.035). Oxytocin receptor concentrations in caruncular endometrium (but not in intercaruncular endometrium) were lower in pregnant than in nonpregnant hinds (P<0.05), but there was no correlation with interferon concentrations in flushings. Corpora luteal concentrations of oxytocin ranged from 1.8 to 51.2 μg/g tissue and declined with advancing blastocyst development. The data are consistent with the hypothesis that sexual dimorphism in trophoblast interferon production leads to differential blastocyst loss and hence to sex ratio skewing on the basis of dominance status.

Role of protein kinase C in the inhibitory action of trophoblast interferons on expression of the oxytocin receptor in sheep endometrium

PhosphoIipid/Ca(2+) -dependent protein kinase C (PKC) and oxytocin receptor were measured in sheep endometrial explants after culture for up to 96 h. Oxytocin receptor binding and PKC activity were reduced by up to 90% in explants exposed to recombinant ovine trophoblast interferon (rolFN-τ), recombinant bovine IFN-α(1) or ovine conceptus secretory proteins (a source of IFN-τ). Inhibition occurred in both caruncular and intercaruncular endometrium taken between days 7 and 10 of the oestrous cycle and in intercaruncular (but not caruncular) endometrium on day 6. Down-regulation of PKC by continued exposure of expiants to 4β-phorbol myristate acetate, or treatment with PKC inhibitors reduced both oxytocin receptor binding and PKC activity by up to 70%. Tyrosine kinase inhibitors were ineffective. Addition of oxytocin or progesterone, which reduce oxytocin receptor bindingin vivo, also lowered oxytocin receptor bindingin vitro in the absence of any effect on PKC. The data indicate that IFN-τ inhibits oxytocin receptor synthesis by a mechanism involving PKC inhibition, but that a non-PKC pathway also operates to control oxytocin receptor binding in non-pregnant animals. These conclusions were supported by measuring PKC activity and oxytocin receptor binding in endometrium without culture. Prolonged exposure of the endometrium to IFN-τin vivo may lead to PKC down regulation by a mechanism analogous to that involved in the action of continuous activation by agonist, and this may represent one function of the prolonged secretion of IFN-τ over a 10-day period in early pregnancy.

Use of repeated biopsies to monitor endometrial oxytocin receptors in the cow

A transcervical biopsy technique was used for the repeated collection of samples of bovine endometrium from individual cows to monitor the development of oxytocin receptors. The collection of biopsies from three cyclic cows at two-day intervals between days 13 and 17 after oestrus did not result in any shortening of the length of the cycle (mean +/- sem 22.0 +/- 0.5 days). Furthermore, the assay of serial blood samples from three cows around the time of biopsy on days 15 and 17 demonstrated that the technique did not induce any release of prostaglandin F2 alpha (PGF2 alpha). Oxytocin receptors were undetectable (< 20 fmol/mg protein) on days 13 and 15 of the cycle, increased to a mean of 121 +/- 16 fmol/mg protein on day 17 (before luteolysis), and were highest in samples collected at oestrus (498 +/- 84 fmol/mg protein). To determine whether a single biopsy of endometrium would give a representative measure of the concentration of uterine oxytocin receptors, the concentrations were determined in samples of caruncular and intercaruncular endometrium collected from the upper, middle and lower proportions of uteri from three oestrous animals at slaughter. The concentrations were similar in both types of tissue (caruncular 730 +/- 15 fmol/mg protein, intercaruncular 727 +/- 48 fmol/mg protein) and there were no differences between the three regions of the uterus.

Oxytocin receptor binding activity in cultured ovine endometrium

Oxytocin receptor binding activity in explants of caruncular and intercaruncular endometrium collected from luteal phase ewes increased during culture. An initial rise in binding activity occurred during the first 24 h of culture in both tissues; binding activity in intercaruncular endometrium continued to increase until day 6, remained unchanged on day 8 and had decreased by day 10 of culture. The maximum concentration of receptors in caruncular endometrium was significantly lower than that in intercaruncular endometrium (P < 0.001) and did not change significantly between days 4 and 10 of culture. Apparent dissociation constants and maximal binding of oxytocin receptor in cultured caruncular and intercaruncular endometrium were 3.09 and 2.72 nmol l-1 and 249 and 459 fmol [3H]oxytocin bound mg-1 protein, respectively. Concentrations of oxytocin receptor remained constant in myometrium during 96 h of culture. The rise in endometrial oxytocin receptor concentration did not result from exposure to fetal calf serum, phenol red or insulin in the culture medium. Substituting fetal calf serum with sheep serum or BSA did not block the rise in receptor binding activity. Actinomycin D and cycloheximide inhibited the rise in receptor concentration in both tissues. Co-culture of lung or kidney with endometrium had no effect on binding activity, whereas co-culture with luteal tissue effectively reduced the rise in oxytocin receptor concentration. To establish whether synthesis of functional oxytocin receptors occurred during culture, the effect of oxytocin on prostaglandin F2 alpha (PGF2 alpha) production was assessed in fresh tissue and after 48 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin receptor concentrations, inositol phosphate turnover and prostaglandin release by endometrium from ewes induced to ovulate during the early post-partum period

Induction of ovulation early post partum in sheep is associated with a high incidence (30-40%) of premature luteolysis. The present study was designed to characterize oxytocin receptor levels, oxytocin-stimulated inositol phosphate (IP) turnover (second messenger) and oxytocin-stimulated prostaglandin F2 alpha (PGF2 alpha) release in the endometrium of post-partum ewes induced to ovulate 21 days after parturition and expected to exhibit a range of corpus luteal functions subsequently. Ovulation was induced on day 21 post partum using a controlled internal drug release device and pregnant mare serum gonadotrophin, and uterine tissues were collected on days 5, 10 or 15 of the cycle (n = 4/day). A further 12 ewes whose interval from previous parturition exceeded 150 days were similarly treated and acted as controls. Measurement of daily peripheral progesterone concentrations revealed that while all control ewes exhibited normal luteal function, abnormal luteal function was evident in two, two and one post-partum ewes studied on days 5, 10 and 15 of the cycle respectively. Oxytocin receptor binding was detected (by receptor-binding assay and in-vitro autoradiography) in the endometrium and myometrium of post-partum ewes at all three stages of the oestrous cycle but only at day 15 in control ewes. To determine IP turnover, 100 mg caruncular endometrium was incubated in duplicate for 2.5 h with 10 microCi [3H]inositol and treated with 0 or 2 mumol oxytocin/l for 30 min, then [3H]inositol mono-, bis- and trisphosphates were quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration of oxytocin receptors in the placenta and fetal membranes of cows during pregnancy and labour.

The concentrations of oxytocin receptors were measured in intercaruncular and caruncular endometrium, fetal cotyledons, chorioallantois and amnion during pregnancy and parturition in cows. Tissues were obtained on days 20 (endometrium only), 50, 100, 150, 200, 225, 250, 275, at term (days 280-284), during labour and within 24 h after calving. Receptor concentrations in intercaruncular endometrium were low on day 20 of pregnancy, 39 +/- 11 fmol mg-1 protein. By day 50, receptor concentrations had increased more than tenfold to 572 +/- 52 fmol and rose steadily until day 250 and then levelled off at about 4500 fmol mg-1. Shortly before parturition, on day 282 +/- 1, a further rise to 7300 +/- 1418 fmol mg-1 was observed, these concentrations were maintained throughout labour. By contrast, caruncular endometrial receptor concentrations remained low until term, mean 145 +/- 15 fmol mg-1, and then rose to 720 +/- 163 fmol mg-1 during labour (cervix 17 cm--fully dilated). Fetal cotyledons and membranes had very low oxytocin receptor concentrations during most of pregnancy, on average only 20 fmol mg-1 protein. At term and during labour, receptor concentrations were significantly increased in both tissues. Mean concentrations during labour were 163 +/- 36 fmol mg-1 for cotyledons, 270 +/- 61 fmol mg-1 for chorioallantois and 311 +/- 121 fmol mg-1 for amnion.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that platelet-activating factor suppresses uterine oxytocin-induced 13,14-dihydro-15-keto-prostaglandin F2 alpha release and phosphatidylinositol hydrolysis in the ewe.

This study was conducted to determine whether platelet-activating factor (PAF) (1) attenuated oxytocin-induced secretion of the prostaglandin (PG) F2 alpha metabolite, PGFM, by the ovine uterus in situ and (2) inhibited the generation of the inositol phosphate secondary messengers by endometrial tissue in response to oxytocin challenge in vitro. Ovariectomized ewes received steroid replacement to mimic the luteal phase. Six ewes received intrauterine injections of 200 micrograms PAF/uterine horn/day on Days 11-15, and 6 ewes were treated with vehicle. All ewes received 1 microgram oxytocin i.v. on Days 13-16. Pretreatment of ewes with PAF significantly suppressed PGFM release in response to oxytocin on Days 14 and 15 (p less than 0.005) compared to vehicle-treated ewes. PAF was not administered on Day 16, and the PGFM response to oxytocin was not different between groups. In a second experiment, ewes were given intrauterine injections of 200 micrograms PAF/uterine horn/day (n = 8) or vehicle (n = 7) on Days 11-15, and all ewes received 1 microgram oxytocin i.v. on Days 13 and 14. On Day 15 the uterus was removed, and the incorporation of 3H-inositol into inositol phosphates was determined in caruncular endometrium. Treatment of ewes with PAF in vivo reduced inositol monophosphate (IP1) generated by oxytocin (10(-6) M) by 56.4%, compared to that in endometrium from vehicle-treated controls, and also inhibited the incorporation of 3H-inositol into glycerophosphoinositol (GPI). If PAF was added to the endometrium during the incubation in vitro, the attenuation of inositol phosphate generation did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of continuous infusion of oxytocin on ovarian function and uterine oxytocin receptor concentration in the cyclic ewe.

Intact cyclic ewes have been used in experiments designed to examine the mechanism by which uterine oxytocin receptor synthesis is controlled during the oestrous cyclic. Previous experiments have shown that the prostaglandin F2 alpha analogue cloprostenol is luteolytic in ewes receiving oxytocin by continuous intra-venous infusion. When ewes receiving oxytocin are given cloprostenol uterine oxytocin receptor concentrations are raised, whereas in animals receiving oxytocin alone, they remain low. To investigate whether inhibition of oxytocin receptor binding activity by oxytocin is either dependent on elevated plasma progesterone concentrations or over-ridden by oestrogens secreted by ovarian follicles maturing as a result of cloprostenol treatment, ewes were given oxytocin by continuous intravenous infusion (3 nmol h-1) between Days 12 and 17 after oestrus and one of the following: no further treatment; cloprostenol [125 micrograms intramuscularly (i.m.)] on Day 15; progesterone, by subcutaneous implant, from Day 14 with cloprostenol on Day 15; medroxyprogesterone acetate (MPA; 6 mg depot i.m.) on Day 14 followed by cloprostenol on Day 15; or oestradiol-17 beta (2.75 mumol i.m.) on Days 14 and 15. Concentrations of oxytocin receptor were measured at autopsy on Day 17 in caruncular endometrium, intercaruncular endometrium and myometrium. Ovarian follicles and corpora lutea were examined to determine the effect of treatment on these tissues. Treatment with oxytocin alone resulted in the maintenance of corpora lutea, reduced follicular development and a low concentration of uterine oxytocin receptor. Cloprostenol initiated luteolysis in oxytocin-treated ewes. This was associated with a high level of oxytocin receptor binding activity in all ewes except those receiving exogenous progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of oestrogen receptors in the endometrium of the ewe

Immunocytochemistry with monoclonal antibodies to the oestrogen receptor (ER) was used to localize ERs in sections of endometrium obtained from cycling and pregnant Corriedale ewes. Representative tissue from Days 4, 10, 14, 15, 16 and 17 of the cycle (Day 0 = onset of oestrus) and Day 15 of pregnancy was used. ER localization was also examined in tissue obtained from ovariectomized (ovex) ewes with and without subcutaneous implants containing oestrogen, progesterone, or oestrogen and progesterone. ER distribution was examined in caruncular endometrium and intercaruncular endometrium. Staining intensity varied according to cell type, stage of the cycle, steroid treatment and pregnancy. No staining was observed in endothelial cells. In all cases, ER was localized within the nuclei of positive cells. Generally, ER levels were high on Day 4 and declined to negligible values by Day 10 (corresponding to peak progesterone values) except in the deep stroma of caruncular endometrium. Positive staining reappeared in stromal cells of caruncles on Day 13 and in the luminal epithelium of intercaruncular tissue on Day 14. Peak intensity was reached on Day 15 for caruncular tissue and Day 16 for intercaruncular tissue. Ovariectomy did not cause an overall reduction in ER levels, whereas treatment with oestrogen and progesterone had variable effects depending on cell type. Progesterone did not suppress overall ER. In Day 15 pregnant tissue, ER was undetectable in all compartments except deep stroma of caruncles, indicating that factors other than progesterone, perhaps embryonic in origin, were responsible. The observation that individual cell types display differential sensitivities to oestrogen and progesterone as regards their expression of ER is consistent with the role of cell-cell interactions as modulators of cellular response to steroids through the oestrous cycle and in pregnancy.